The overall commercial aim of this project is to create new ketolide antiinfective agents using combinatorial biosynthesis technologies. The specific aims are: l) to develop a versatile plasmid-host expression system in Streptomyces venezuelae through which pikromycin analogues can be generated rapidly and systematically; and 2) generate novel ketolides by creating a 'hybrid biosynthetic pathway that combines the pikromycin polyketide synthase (PikA) with a cephamycin C non-ribosomal peptide synthetase domain. A key aspect of the proposed program involves refining a versatile expression system for engineered biosynthesis of complex polyketide natural products. This will provide plasmid- or chromosome-directed biosynthesis of the polyketide derived and aminosugar derived portions of the ketolide molecules. The development of this versatile set of molecular genetic tools will enable rapid generation of large numbers of ketolide structures for lead discovery of new antiinfective agents. Concurrent to the development of our molecular genetic tools, two specific hybrid biosynthetic pathways will be generated that introduce versatile molecular handles for creation of novel ketolide antibiotics. We expect this technology will provide a rapid source of novel ketolide molecules and structural templates for analysis in antimicrobial assay systems, and for further development using combinatorial chemistry or medicinal chemistry approaches. PROPOSED COMMERCIAL APPLICATIONS: The proposed research involves the development and reduction to practice of a technology that will provide rapid creation of new chemical entities for generation of novel pharmaceuticals with activity against drug-resistant pathogenic bacteria.